QTL Determining Diel Flight Activity in Male Culex pipiens Mosquitoes.

Members of the Culex pipiens complex differ in physiological traits that facilitate their survival in diverse environments. Assortative mating within the complex occurs in some regions where autogenous (the ability to lay a batch of eggs without a blood meal) and anautogenous populations are sympatric, and differences in mating behaviors may be involved. For example, anautogenous populations mate in flight/swarms, while autogenous populations often mate at rest. Here, we characterized flight activity of males and found that anautogenous strain males were crepuscular, while autogenous strain males were crepuscular and nocturnal, with earlier activity onset times. We conducted quantitative trait locus (QTL) mapping to explore the genetic basis of circadian chronotype (crepuscular vs. crepuscular and nocturnal) and time of activity onset. One major-effect QTL was identified for chronotype, while 3 QTLs were identified for activity onset. The highest logarithm of the odds (LOD) score for the chronotype QTL coincides with a chromosome 3 marker that contains a 15-nucleotide indel within the coding region of the canonical clock gene, cryptochrome 2. Sequencing of this locus in 7 different strains showed that the C-terminus of CRY2 in the autogenous forms contain deletions not found in the anautogenous forms. Consequently, we monitored activity in constant darkness and found males from the anautogenous strain exhibited free running periods of ~24 h while those from the autogenous strain were ~22 h. This study provides novel insights into the genetic basis of flight behaviors that likely reflect adaptation to their distinct ecological niches.

The making of a pest: Insights from the evolution of chemosensory receptor families in a pestiferous and invasive fly, Drosophila suzukii.

BACKGROUND: Drosophila suzukii differs from other melanogaster group members in their proclivity for laying eggs in fresh fruit rather than in fermenting fruits. Olfaction and gustation play a critical role during insect niche formation, and these senses are largely mediated by two important receptor families: olfactory and gustatory receptors (Ors and Grs). Earlier work from our laboratory has revealed how the olfactory landscape of D. suzukii is dominated by volatiles derived from its unique niche. Signaling and reception evolve in synchrony, since the interaction of ligands and receptors together mediate the chemosensory behavior. Here, we manually annotated the Ors and Grs in D. suzukii and two close relatives, D. biarmipes and D. takahashii, and compared these repertoires to those in other melanogaster group drosophilids to identify candidate chemoreceptors associated with D. suzukii's unusual niche utilization. RESULTS: Our comprehensive annotations of the chemosensory genomes in three species, and comparative analysis with other melanogaster group members provide insights into the evolution of chemosensation in the pestiferous D. suzukii. We annotated a total of 71 Or genes in D. suzukii, with nine of those being pseudogenes (12.7 %). Alternative splicing of two genes brings the total to 62 genes encoding 66 Ors. Duplications of Or23a and Or67a expanded D. suzukii's Or repertoire, while pseudogenization of Or74a, Or85a, and Or98b reduced the number of functional Ors to roughly the same as other annotated species in the melanogaster group. Seventy-one intact Gr genes and three pseudogenes were annotated in D. suzukii. Alternative splicing in three genes brings the total number of Grs to 81. We identified signatures of positive selection in two Ors and three Grs at nodes leading to D. suzukii, while three copies in the largest expanded Or lineage, Or67a, also showed signs of positive selection at the external nodes. CONCLUSION: Our analysis of D. suzukii's chemoreceptor repertoires in the context of nine melanogaster group drosophilids, including two of its closest relatives (D. biarmipes and D. takahashii), revealed several candidate receptors associated with the adaptation of D. suzukii to its unique ecological niche.

Whole transcriptome responses among females of the filariasis and arbovirus vector mosquito Culex pipiens implicate TGF-ß signaling and chromatin modification as key drivers of diapause induction.

Culex pipiens mosquitoes are important disease vectors inhabiting temperate zones, worldwide. The seasonal reduction in temperature and photoperiod accompanying late summer and early fall prompts female mosquitoes to enter diapause, a stage of developmental arrest and physiological conditioning that enhances survival during the winter months. To investigate the molecular mechanisms underlying diapause induction, we used custom whole transcriptome microarrays to identify differences in gene expression following exposure to nondiapause (long days, 25 °C) and diapause-inducing (short days, 18 °C) environmental conditions. Using a two-way ANOVA, we identified 1130 genes that were differentially expressed. We used the expression of these genes across three time points to construct a gene co-expression network comprising five modules. Genes in modules 1, 2, and 3 were largely up-regulated, while genes in modules 4 and 5 were down-regulated when compared to nondiapause conditions. Pathway enrichment analysis of the network modules revealed some potential regulatory mechanisms driving diapause induction. Module 1 was enriched for genes in the TGF-ß and Wnt signaling pathways; module 2 was enriched for genes involved in insect hormone biosynthesis, specifically, ecdysone synthesis; module 3 was enriched for genes involved in chromatin modification; and module 5 was enriched for genes in the circadian rhythm pathway. Our results suggest that TGF-ß signaling and chromatin modification are key drivers for the integration of environmental signals into the diapause induction phase in C. pipiens mosquitoes.

An alternative chicken-based diet for mass-rearing screwworm flies.

Screwworm flies are mass-reared and released along the Panama-Colombia border to prevent reinfestation of Central and North America. The cost of the production facility, labor, and diet materials makes mass-rearing the most expensive component of the program. The mass-rearing diet has a large impact on the quality and quantity of insects produced, both of which are necessary for the successful implementation of the sterile insect technique. The diet currently used to rear screwworm flies in Panama contains dried bovine red blood cells, dried bovine plasma, egg powder, milk replacement powder, cellulose (thickening agent), formaldehyde (antimicrobial), and water. Here, we tested an alternative diet containing 2 chicken by-products, which cost less and are locally available, to replace the egg powder and milk replacement powder currently used in the diet. We used 2 screwworm colony strains in our test, the current production strain (Jamaica) and an early female-lethal strain. The chicken diet performed similarly to the production diet with the Jamaica strain, while further optimization will likely be needed for transgenic strain. Finally, nutritional analysis conducted on 7 diet ingredients will assist with diet optimization and the identification of alternative diet ingredients.

Antennal transcriptome analysis reveals sensory receptors potentially associated with host detection in the livestock pest Lucilia cuprina.

BACKGROUND: Lucilia cuprina (Wiedemann, 1830) (Diptera: Calliphoridae) is the main causative agent of flystrike of sheep in Australia and New Zealand. Female flies lay eggs in an open wound or natural orifice, and the developing larvae eat the host's tissues, a condition called myiasis. To improve our understanding of host-seeking behavior, we quantified gene expression in male and female antennae based on their behavior. METHODS: A spatial olfactometer was used to evaluate the olfactory response of L. cuprina mated males and gravid females to fresh or rotting beef. Antennal RNA-Seq analysis was used to identify sensory receptors differentially expressed between groups. RESULTS: Lucilia cuprina females were more attracted to rotten compared to fresh beef (> fivefold increase). However, males and some females did not respond to either type of beef. RNA-Seq analysis was performed on antennae dissected from attracted females, non-attracted females and males. Transcripts encoding sensory receptors from 11 gene families were identified above a threshold (≥ 5 transcript per million) including 49 ATP-binding cassette transporters (ABCs), two ammonium transporters (AMTs), 37 odorant receptors (ORs), 16 ionotropic receptors (IRs), 5 gustatory receptors (GRs), 22 odorant-binding proteins (OBPs), 9 CD36-sensory neuron membrane proteins (CD36/SNMPs), 4 chemosensory proteins (CSPs), 4 myeloid lipid-recognition (ML) and Niemann-Pick C2 disease proteins (ML/NPC2), 2 pickpocket receptors (PPKs) and 3 transient receptor potential channels (TRPs). Differential expression analyses identified sex-biased sensory receptors. CONCLUSIONS: We identified sensory receptors that were differentially expressed between the antennae of both sexes and hence may be associated with host detection by female flies. The most promising for future investigations were as follows: an odorant receptor (LcupOR46) which is female-biased in L. cuprina and Cochliomyia hominivorax Coquerel, 1858; an ABC transporter (ABC G23.1) that was the sole sensory receptor upregulated in the antennae of females attracted to rotting beef compared to non-attracted females; a female-biased ammonia transporter (AMT_Rh50), which was previously associated with ammonium detection in Drosophila melanogaster Meigen, 1830. This is the first report suggesting a possible role for ABC transporters in L. cuprina olfaction and potentially in other insects.

Composite linkage map and enhanced genome map for Culex pipiens complex mosquitoes.

We report here the development of 65 novel microsatellite loci and construction of a composite genetic linkage map for Culex pipiens complex mosquitoes. Microsatellites were identified by in silico screening of the Culex quinquefasciatus genome assembly. Cross-species utility of 73 microsatellites for population studies in C. pipiens sensu stricto and C. quinquefasciatus was evaluated by genotyping a subset of samples collected in Indiana, United States, and Point Fortin, Trinidad. Allele frequencies of 67 microsatellites were within Hardy-Weinberg expectations in both population subsets. A composite linkage map was constructed based on restriction fragment length polymorphism and microsatellite polymorphisms in 12 independent F1 intercross mapping populations. The composite map consists of 61 marker loci totaling 183.9 cM distributed across the 3 linkage groups. These loci cover 29.5, 88.8, and 65.6 cM on chromosomes I-III, respectively, and allow for assignment of 10.4% of the genome assembly and 13.5% of the protein coding genes to chromosome position. Our results suggest that these microsatellites will be useful for mapping and population studies of 2 pervasive species in the C. pipiens complex. Moreover, the composite map presented here will serve as a basis for the construction of high-resolution genetic and physical maps, as well as detection of quantitative trait loci to aid in the investigation of complex genetic traits influencing phenotypes of interest.

A new formulation of screwworm fly attractant with reduced hazardous chemicals and transport restrictions.

New World screwworm flies, Cochliomyia hominivorax (Coquerel), are obligate parasites of warm-blooded animals. They were eradicated from North and Central America during the mid-20th to early-21st centuries using the sterile insect technique (SIT), a method presently employed to maintain a permanent barrier between Central and South America. Lures are an important component of the screwworm eradication program, where they are used for surveillance, sample collection, and strain evaluation in the field. The first chemical lure, later named swormlure, was developed based on the attractiveness of C. hominivorax to volatile organic compounds (VOCs) produced from decomposing animal tissues. The formulation has changed little over the years and presently contains 10 chemicals, one of which is dimethyl disulfide (DMDS). Restrictions on the transport of DMDS have recently impeded its use in swormlure-4 (SL-4). However, dimethyl trisulfide (DMTS) is not as highly restricted and can be shipped via air transportation. Both chemicals are produced by microbial decomposition of animal tissues. Here, we conducted field trials using three releases of sterile C. hominivorax, each comprising approximately 93,000 flies, to test the efficacy of SL-4, containing DMDS, to swormlure-5 (SL-5) containing DMTS. Traps baited with SL-4 and SL-5 captured 575 (mean = 191.7, SD 17.9) and 665 (mean = 221.7, SD 33.2) C. hominivorax, respectively (df = 19, F = 1.294, P = 0.269). However, traps baited with SL-5 captured considerably more Cochliomyia macellaria (Fabricius), a closely related but nontarget fly.

Genomic analysis of two phlebotomine sand fly vectors of Leishmania from the New and Old World.

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.

The microbiome of wild and mass-reared new world screwworm, Cochliomyia hominivorax.

Insect population control through continual releases of large numbers of sterile insects, called sterile insect technique (SIT), is only possible if one can mass-rear large quantities of healthy insects. Adaptation of insect stocks to rearing conditions and artificial feeding systems can have a multitude of negative effects such as inbreeding depression, reduced compatibility with wild strains, unintentional selection for traits that lower fitness after release, and an altered microbiome. Changes to insect microbiomes can have many effects on insects ranging from a reduction in sex pheromones or reduced fitness. Thus understanding these systems is important for mass rearing and the performance of the sterile insect control programs. In this study we explored the microbiome of the New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) an economically important parasite of warm-blooded animals. Samples from myiases in cows and wild adults were compared to and mass-reared flies used by the SIT program. Significant differences were observed between these treatments, with wild captured flies having a significantly more diverse microbial composition. Bacteria known to stimulate oviposition were found in both wild and mass-reared flies. Two bacteria of veterinary importance were abundant in wild flies, suggesting screwworm is a potential vector of these diseases. Overall, this study provides the screwworm eradication program a platform to continue exploring the effects associated bacteria have on screwworm fitness.

Quantitative Trait Locus Determining the Time of Blood Feeding in Culex pipiens (Diptera: Culicidae).

Mosquitoes and other blood feeding arthropods are vectors of pathogens causing serious human diseases, such as Plasmodium spp. (malaria), Wuchereria bancrofti (lymphatic filariasis), Borrelia burgdorferi (Lyme disease), and viruses causing dengue, Zika, West Nile, chikungunya, and yellow fever. Among the most effective strategies for the prevention of vector-borne diseases are those aimed at reducing human-vector interactions, such as insecticide applications and insecticide-treated bed nets (ITNs). In some areas where ITNs are widely used, behavioral adaptations have resulted in mosquitoes shifting their time of blood feeding to earlier or later in the night when the bed nets are not being employed. Little is known about the genetic basis of these behavioral shifts. We conducted quantitative trait locus (QTL) analysis using two strains of Culex pipiens sensu lato with contrasting blood feeding behaviors, wherein the lab adapted Shasta strain blood feeds at any time of the day or night, while the newly established Trinidad strain feeds only at night. We identified a single locus on chromosome 2 associated with the observed variation in feeding times. None of the core clock genes period, timeless, clock, cycle, PAR-domain protein 1, vrille, discs overgrown, cryptochrome 1, or cryptochrome 2 were located within the QTL region. We then monitored locomotor behavior to determine if they differed in their flight activity. The highly nocturnal Trinidad strain showed little daytime activity while the day-feeding Shasta strain was active during the day, suggesting blood feeding behavior and flight activity are physiologically linked.

Phylogenomics of Tick Inward Rectifier Potassium Channels and Their Potential as Targets to Innovate Control Technologies.

This study was conducted to enhance the identification of novel targets to develop acaricides that can be used to advance integrated tick-borne disease management. Drivers for the emergence and re-emergence of tick-borne diseases affecting humans, livestock, and other domestic animals in many parts of the world include the increased abundance and expanded geographic distribution of tick species that vector pathogens. The evolution of resistance to acaricides among some of the most important tick vector species highlights the vulnerability of relying on chemical treatments for tick control to mitigate the health burden of tick-borne diseases. The involvement of inward rectifier potassium (Kir) channels in homeostasis, diuresis, and salivary gland secretion in ticks and other pests identified them as attractive targets to develop novel acaricides. However, few studies exist on the molecular characteristics of Kir channels in ticks. This bioinformatic analysis described Kir channels in 20 species of hard and soft ticks. Summarizing relevant investigations on Kir channel function in invertebrate pests allowed the phylogenomic study of this class of ion channels in ticks. How this information can be adapted to innovate tick control technologies is discussed.

New World screwworm (Cochliomyia hominivorax) myiasis in feral swine of Uruguay: One Health and transboundary disease implications.

BACKGROUND: Feral swine (Sus scrofa) are highly invasive and threaten animal and human health in the Americas. The screwworm (Cochliomyia hominivorax) is listed by the World Organization for Animal Health as a notifiable infestation because myiasis cases affect livestock, wildlife, and humans in endemic areas, and outbreaks can have major socioeconomic consequences in regions where the screwworm has been eradicated. However, a knowledge gap exists on screwworm infestation of feral swine in South America, where the screwworm is endemic. Here, we report screwworm infestation of feral swine harvested in Artigas Department (Uruguay), where the Republic of Uruguay shares borders with Brazil and Argentina. METHODS: Myiasis caused by the larvae of screwworm were identified in feral swine with the support and collaboration of members of a local feral swine hunting club over a 3-year period in the Department of Artigas. Harvested feral swine were examined for the presence of lesions where maggots causing the myiasis could be sampled and processed for taxonomic identification. The sites of myiasis on the body of infested feral swine and geospatial data for each case were recorded. The sex and relative size of each feral swine were also recorded. Temperature and precipitation profiles for the region were obtained from public sources. RESULTS: Myiases caused by screwworms were recorded in 27 of 618 the feral swine harvested. Cases detected in males weighing > 40 kg were associated with wounds that, due to their location, were likely caused by aggressive dominance behavior between adult males. The overall prevalence of screwworm infestation in the harvested feral swine was associated with ambient temperature, but not precipitation. Case numbers peaked in the warmer spring and summer months. CONCLUSIONS: This is the first report on myiasis in feral swine caused by screwworm in South America. In contrast to myiasis in cattle, which can reach deep into host tissues, screwworms in feral swine tended to cause superficial infestation. The presence of feral swine in screwworm endemic areas represents a challenge to screwworm management in those areas. Screwworm populations maintained by feral swine may contribute to human cases in rural areas of Uruguay, which highlights the importance of the One Health approach to the study of this invasive host species-ectoparasite interaction.

Molecular signatures of sexual communication in the phlebotomine sand flies.

Phlebotomine sand flies employ an elaborate system of pheromone communication wherein males produce pheromones that attract other males to leks (thus acting as an aggregation pheromone) and females to the lekking males (sex pheromone). In addition, the type of pheromone produced varies among populations. Despite the numerous studies on sand fly chemical communication, little is known of their chemosensory genome. Chemoreceptors interact with chemicals in an organism's environment to elicit essential behaviors such as the identification of suitable mates and food sources. Thus, they play important roles during adaptation and speciation. Major chemoreceptor gene families, odorant receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs) together detect and discriminate the chemical landscape. Here, we annotated the chemoreceptor repertoire in the genomes of Lutzomyia longipalpis and Phlebotomus papatasi, major phlebotomine vectors in the New World and Old World, respectively. Comparison with other sequenced Diptera revealed a large and unique expansion where over 80% of the ~140 ORs belong to a single, taxonomically restricted clade. We next conducted a comprehensive analysis of the chemoreceptors in 63 L. longipalpis individuals from four different locations in Brazil representing allopatric and sympatric populations and three sex-aggregation pheromone types (chemotypes). Population structure based on single nucleotide polymorphisms (SNPs) and gene copy number in the chemoreceptors corresponded with their putative chemotypes, and corroborate previous studies that identified multiple populations. Our work provides genomic insights into the underlying behavioral evolution of sexual communication in the L. longipalpis species complex in Brazil, and highlights the importance of accounting for the ongoing speciation in central and South American Lutzomyia that could have important implications for vectorial capacity.

Physiological and molecular correlates of the screwworm fly attraction to wound and animal odors.

The screwworm fly, Cochliomyia hominivorax (Coquerel), was successfully eradicated from the United States by the sterile insect technique (SIT). However, recent detection of these flies in the Florida Keys, and increased risk of introductions to the other areas warrant novel tools for management of the flies. Surveillance, a key component of screwworm control programs, utilizes traps baited with rotting liver or a blend of synthetic chemicals such as swormlure-4. In this work, we evaluated the olfactory physiology of the screwworm fly and compared it with the non-obligate ectoparasitic secondary screwworm flies, C. macellaria, that invade necrotic wound and feed on dead tissue. These two species occur in geographically overlapping regions. C. macellaria, along with other blowflies such as the exotic C. megacephala, greatly outnumber C. hominivorax in the existing monitoring traps. Olfactory responses to swormlure-4 constituents between sex and mating status (mated vs unmated) in both species were recorded and compared. Overall, responses measured by the antennograms offered insights into the comparative olfactory physiology of the two fly species. We also present detailed analyses of the antennal transcriptome by RNA-Sequencing that reveal significant differences between male and female screwworm flies. The differential expression patterns were confirmed by quantitative PCR. Taken together, this integrated study provides insights into the physiological and molecular correlates of the screwworm's attraction to wounds, and identifies molecular targets that will aid in the development of odorant-based fly management strategies.

Genomic analyses of a livestock pest, the New World screwworm, find potential targets for genetic control programs.

The New World Screwworm fly, Cochliomyia hominivorax, is a major pest of livestock in South America and Caribbean. However, few genomic resources have been available for this species. A genome of 534 Mb was assembled from long read PacBio DNA sequencing of DNA from a highly inbred strain. Analysis of molecular evolution identified 40 genes that are likely under positive selection. Developmental RNA-seq analysis identified specific genes associated with each stage. We identify and analyze the expression of genes that are likely important for host-seeking behavior (chemosensory), development of larvae in open wounds in warm-blooded animals (heat shock protein, immune response) and for building transgenic strains for genetic control programs including gene drive (sex determination, germline). This study will underpin future experiments aimed at understanding the parasitic lifestyle of the screwworm fly and greatly facilitate future development of strains for efficient systems for genetic control of screwworm.

Genome annotation improvements from cross-phyla proteogenomics and time-of-day differences in malaria mosquito proteins using untargeted quantitative proteomics.

The malaria mosquito, Anopheles stephensi, and other mosquitoes modulate their biology to match the time-of-day. In the present work, we used a non-hypothesis driven approach (untargeted proteomics) to identify proteins in mosquito tissue, and then quantified the relative abundance of the identified proteins from An. stephensi bodies. Using these quantified protein levels, we then analyzed the data for proteins that were only detectable at certain times-of-the day, highlighting the need to consider time-of-day in experimental design. Further, we extended our time-of-day analysis to look for proteins which cycle in a rhythmic 24-hour ("circadian") manner, identifying 31 rhythmic proteins. Finally, to maximize the utility of our data, we performed a proteogenomic analysis to improve the genome annotation of An. stephensi. We compare peptides that were detected using mass spectrometry but are 'missing' from the An. stephensi predicted proteome, to reference proteomes from 38 other primarily human disease vector species. We found 239 such peptide matches and reveal that genome annotation can be improved using proteogenomic analysis from taxonomically diverse reference proteomes. Examination of 'missing' peptides revealed reading frame errors, errors in gene-calling, overlapping gene models, and suspected gaps in the genome assembly.

Evidence for an Overwintering Population of Aedes aegypti in Capitol Hill Neighborhood, Washington, DC.

Aedes aegypti is an invasive, highly anthropophilic mosquito and a major vector for dengue and chikungunya. Population persistence in the continental United States is reportedly limited to southward of the average 10°C winter isotherm, which in the east, bisects Alabama, Mississippi, Georgia, and South Carolina. We report on summer collections and genotypic analyses of Ae. aegypti collected in the Capitol Hill neighborhood in Washington, DC (WDC). Analysis of a 441-bp fragment of the mitochondrial cytochrome oxidase I gene sequence identified the same two haplotype sequences during 2011-2014, and placed these within two discrete groups known to be derived from lineages resident in the Americas. Analysis of 10 microsatellite loci for specimens collected during 2011-2014 revealed no evidence for introgression of new alleles across years. Overall, our data support a conclusion that this represents a resident WDC population, likely maintained during winter months in a subterranean habitat that facilitates year-round survival.

Enhancing genome investigations in the mosquito Culex quinquefasciatus via BAC library construction and characterization.

BACKGROUND: Culex quinquefasciatus (Say) is a major species in the Culex pipiens complex and an important vector for several human pathogens including West Nile virus and parasitic filarial nematodes causing lymphatic filariasis. It is common throughout tropical and subtropical regions and is among the most geographically widespread mosquito species. Although the complete genome sequence is now available, additional genomic tools are needed to improve the sequence assembly. FINDINGS: We constructed a bacterial artificial chromosome (BAC) library using the pIndigoBAC536 vector and HindIII partially digested DNA isolated from Cx. quinquefasciatus pupae, Johannesburg strain (NDJ). Insert size was estimated by NotI digestion and pulsed-field gel electrophoresis of 82 randomly selected clones. To estimate genome coverage, each 384-well plate was pooled for screening with 29 simple sequence repeat (SSR) and five gene markers. The NDJ library consists of 55,296 clones arrayed in 144 384-well microplates. Fragment insert size ranged from 50 to 190 kb in length (mean = 106 kb). Based on a mean insert size of 106 kb and a genome size of 579 Mbp, the BAC library provides ~10.1-fold coverage of the Cx. quinquefasciatus genome. PCR screening of BAC DNA plate pools for SSR loci from the genetic linkage map and for four genes associated with reproductive diapause in Culex pipiens resulted in a mean of 9.0 positive plate pools per locus. CONCLUSION: The NDJ library represents an excellent resource for genome assembly enhancement and characterization in Culex pipiens complex mosquitoes.

Genome-based microsatellite development in the Culex pipiens complex and comparative microsatellite frequency with Aedes aegypti and Anopheles gambiae.

BACKGROUND: Mosquitoes in the Culex pipiens complex are among the most medically important vectors for human disease worldwide and include major vectors for lymphatic filariasis and West Nile virus transmission. However, detailed genetic studies in the complex are limited by the number of genetic markers available. Here, we describe methods for the rapid and efficient identification and development of single locus, highly polymorphic microsatellite markers for Cx. pipiens complex mosquitoes via in silico screening of the Cx. quinquefasciatus genome sequence. METHODOLOGY/PRINCIPAL FINDINGS: Six lab colonies representing four Cx. pipiens and two Cx. quinquefasciatus populations were utilized for preliminary assessment of 38 putative loci identified within 16 Cx. quinquefasciatus supercontig assemblies (CpipJ1) containing previously mapped genetic marker sequences. We identified and validated 12 new microsatellite markers distributed across all three linkage groups that amplify consistently among strains representing the complex. We also developed four groups of 3-5 microsatellite loci each for multiplex-ready PCR. Field collections from three cities in Indiana were used to assess the multiplex groups for their application to natural populations. All were highly polymorphic (Mean  = 13.0 alleles) per locus and reflected high polymorphism information content (PIC) (Mean  = 0.701). Pairwise F(ST) indicated population structuring between Terre Haute and Fort Wayne and between Terre Haute and Indianapolis, but not between Fort Wayne and Indianapolis. In addition, we performed whole genome comparisons of microsatellite motifs and abundance between Cx. quinquefasciatus and the primary vectors for dengue virus and malaria parasites, Aedes aegypti and Anopheles gambiae, respectively. CONCLUSIONS/SIGNIFICANCE: We demonstrate a systematic approach for isolation and validation of microsatellites for the Cx. pipiens complex by direct screen of the Cx. quinquefasciatus genome supercontig assemblies. The genome density of microsatellites is greater in Cx. quinquefasciatus (0.26%) than in Ae. aegypti (0.14%), but considerably lower than in An. gambiae (0.77%).